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Viral particles are great gene delivery vehicles for your expression experiments! But each vehicle is engineered to drive best over certain terrains or have a certain amount of trunk space. For example, some viral particles, like the Baculovirus, are like container trucks that can carry large DNA loads but can only “drive” into limited mammalian cell types. Other viral particles, like the AAV, are like sports cars that can “drive” into most cell types; however, its small trunk space means it can only carry small DNA loads. With this in mind, how do you know which gene delivery vehicle “model” is right for your project? This article will be your model comparison guide - whether you’re over-expressing, knocking down, knocking out, or knocking in a gene, there’s a viral expression system perfect for you!
Viral transduction is the process of introducing foreign nucleic acids into cells. This technique can be used to modify gene expression levels, reprogram stem cells, generate stable cell lines and more! Viral transduction can be used for both in vivo or in vitro applications, making it one of the most versatile gene delivery tools available to life science researchers. Read more about viral transduction.
Safety
To engineer viral particles, the original pathogenic virus is modified to delete the genes involved in viral replication or packaging, while preserving the virus’s ability to infect and deliver genes to the target cell. The resulting recombinant viruses are safe to handle as they are not able to replicate uncontrollably without helper viruses or specialized cell lines that provide these required, deleted genes. Most commercially available recombinant viruses are replication-incompetent. When it comes to the use of recombinant viruses in gene therapy, one safety concern is that the DNA insert that is introduced may disrupt a tumor-suppressor gene or activate an oncogene that will cause the target cell to become cancerous.
Minimal cytotoxicity
For in vivo studies, it is important to use recombinant viruses that have little to no effect on cell physiology. Non-immunogenic recombinant viruses such as AAV will not be recognized as a foreign invader, and therefore will not incite an immune response in the host organism.
Genetic stability
Recombinant viruses are typically selected for their ability to deliver DNA into a host cell in a stable and reliable manner so that experimental results are reproducible. For many viruses, stability decreases as the size of the DNA insert increases. This is due to the increasing risk of genomic rearrangement in larger DNA fragments that lead to loss of the inserted sequence or even parts of the viral genome itself.
Broad or cell specific targeting
Recombinant viruses can be engineered to target a specific cell type or a wide range of cell types by modifying the viral envelope proteins. This process is called “pseudotyping” and involves introducing viral envelope proteins from one virus to another in order to alter what cell types the virus is able to target (called “ host tropism”). This allows flexibility in the experiments that can be performed. For instance, VSV-G, a glycoprotein from Vesicular stomatitis virus, is widely used to pseudotype lentiviral vectors as a way to broaden host tropism and increase infectivity.
Ability to use selection markers (GFP, antibiotic resistant markers)
Selection markers can be incorporated into viral vectors to help identify and isolate cells that have been successfully transduced. Some commonly used markers are antibiotic resistance markers, fluorescent reporters (e.g., GFP), or the lacZa sequence containing an internal multiple cloning site (used for blue-white colony screening).
Depending on your application, you may select a recombinant virus based on its ability to offer transient or stable expression of the delivered DNA.
Transient gene expression systems
In transient expression systems, such as the recombinant adenovirus, the foreign genetic material is delivered into the host cell but does not become integrated into the cell’s genome and is therefore not passed to daughter cells. Transiently infected cells will only express the foreign genetic materials for a short period of time (usually several days) before being degraded, with expression eventually lost in subsequent cycles of cell division. This expression type is suitable for applications where you want rapid gene expression, small-scale protein production, or short term gene expression studies such as gene knockdowns/silencing with RNAi (changes in gene expression will be observable 1-2 days post transduction).
Stable gene expression systems
In stable expression systems, such as the recombinant lentivirus, the foreign genetic material is integrated into the host cell’s genome and will be replicated and passed to future daughter cells. All subsequent generations of the cell will express the foreign gene indefinitely, becoming what is termed a “stable cell line”. Due to the random nature of genetic integrations, such cells will often need to be further selected to isolate a single “clone” with the same genetic modification/expression profile. This expression type is suitable for applications where you want larger scale protein production, for long term gene expression studies, or for the development of gene therapies.
Refer to the table below to find which viral expression system would be the most compatible for your experiment based on packaging capacity, transduction efficiency, cell types it can infect, expression type, integration ability, host immune response, and the type of genetic material that can be delivered. This table includes the most widely used viral vector systems.
| Characteristic | Lentivirus | Adenovirus | Adeno-Associated virus | Retrovirus | Herpes Simplex Virus (HSV) | Baculovirus |
| Packaging Capacity | 8 kb | 8 kb | 4.7 kb | 8 kb | Theoretically up to 150 kb | Theoretically > 100 kb |
| Transduction Efficiency | Most Dividing/Non-dividing Cells | Most Dividing/Non-dividing Cells (with high transduction rate towards Primary Cells | All Cell Types (depending on Serotype) | Dividing Cells | Most Dividing/Non-dividing Cells (ideal for Neuronal Cells) | Most Dividing/Non-dividing Cells (including Bacterial, |
| Infection | ●●● | ●●● | ●●● | ●●● | ●●● | ●●● |
| Expression | Stable | Transient | Transient or Stable | Stable | Transient | Transient or Stable |
| Integrating | Yes | No | Site-specific Integration | Yes | No (but may replicate separately from the host) | No |
| Immune Response | ●●● | ●●●● | ●● | ●●●● | ●●●● | ●●● |
| Genetic Material | RNA | Double Stranded Linear DNA | Single Stranded Linear DNA | RNA | Double Stranded Linear DNA | Double Stranded Linear DNA |
Lentivirus: A subtype of retroviruses, the recombinant lentivirus is the workhorse of many labs. Due to its ability to stably integrate DNA inserts into both dividing and non-dividing cells, it is commonly used to generate stable cell lines, deliver CRISPR systems, and more.
Adenovirus: Because adenoviruses have high transduction efficiencies and elicit strong immune responses in vivo, they are ideal platforms for vaccine production and gene therapy development
Adeno-associated Virus (AAV): AAV have the unique ability to target specific tissue types without triggering a severe immune response in vivo, making it useful for developing targeted cancer or gene therapies.
Retrovirus: Recombinant retroviruses are one of the oldest tools for gene delivery and have been successfully used to stably integrate genes into haematopoietic stem cells in vivo.
Herpes Simplex Virus (HSV): Due to its non-integrative nature and ability to package large insert sizes, HSV has been successfully used to deliver large DNA inserts into neuronal cells for gene expression.
Baculovirus: Because it can infect insect cells which are easier to culture and can preserve post-translational modifications, baculoviruses are typically used for large scale production of proteins, especially glycoproteins or membrane proteins.
Are you trying to express a gene or an RNAi? How big is your expression cassette? Are you looking for long-term or transient expression? What kind of cells are you trying to infect? These are some of the questions you’ll need to answer in order to determine which viral expression system is best for your project! Feeling overwhelmed? Not to worry! Our fun and interactive “Vector Selection Tool” will guide you through each of these questions and suggest which vector system would be the most ideal for your experiment. Get simple, clear recommendations for every application! Try our Vector Selection Tool.
abm also offers a custom cloning service as well as our “Plasmid Planner Tool” that you can use to easily build your dream vector. You may use our Plasmid Planner Tool to choose your own promoters, inserts, tags and even antibiotic resistance markers!
